Dominant frontonasal dysplasia with ectodermal defects results from increased activity of ALX4.

Frontonasal dysplasia (FND) refers to a group of rare developmental disorders characterized by abnormal morphology of the craniofacial region. We studied a family manifesting with clinical features typical for FND2 including neurobehavioral abnormalities, hypotrichosis, hypodontia, and facial dysmorphism. Whole-exome sequencing analysis identified a novel heterozygous frameshift insertion in ALX4 (c.985_986insGTGC, p.Pro329Argfs*115), encoding aristaless homeobox 4. This and a previously reported dominant FND2-causing variant are predicted to result in the formation of a similar abnormally elongated protein tail domain. Using a reporter assay, we showed that the elongated ALX4 displays increased activity. ALX4 negatively regulates the Wnt/ß-catenin pathway and accordingly, patient keratinocytes showed altered expression of genes associated with the WNT/ß-catenin pathway, which in turn may underlie ectodermal manifestations in FND2. In conclusion, dominant FND2 with ectodermal dysplasia results from frameshift variants in ALX4 exerting a gain-of-function effect.

Vorinostat, a histone deacetylase inhibitor, as a potential novel treatment for psoriasis.

BACKGROUND: Psoriasis is characterized by aberrant activation of several pro-inflammatory circuits as well as abnormal hyperproliferation and dysregulated apoptosis of keratinocytes (KCs). Most currently available therapeutic options primarily target psoriasis-associated immunological defects rather than epidermal abnormalities. OBJECTIVE: To investigate the efficacy of the histone deacetylase (HDAC) inhibitor, Vorinostat, in targeting hyperproliferation and impaired apoptosis in psoriatic skin. METHODS: Vorinostat effect was investigated in primary KCs cell cultures using cell cycle analysis by flow cytometry, apoptosis assays (Annexin V-FICH and caspase-3/7) and antibody arrays, qRT-PCR and immunohistochemistry. Vorinostat impact on clinical manifestations of psoriasis was investigated in a chimeric mouse model. RESULTS: Vorinostat was found to inhibit KCs proliferation and to induce their differentiation and apoptosis. Using a chimeric mouse model, vorinostat was found to result in marked attenuation of a psoriasiform phenotype with a significant decrease in epidermal thickness and inhibition of epidermal proliferation. CONCLUSIONS: Our results support the notion that vorinostat, a prototypic HDAC inhibitor, may be of potential use in the treatment of psoriasis and other hyperproliferative skin disorders.

The Role of Desmoglein 1 in Gap Junction Turnover Revealed through the Study of SAM Syndrome.

An effective epidermal barrier requires structural and functional integration of adherens junctions, tight junctions, gap junctions (GJ), and desmosomes. Desmosomes govern epidermal integrity while GJs facilitate small molecule transfer across cell membranes. Some patients with severe dermatitis, multiple allergies, and metabolic wasting (SAM) syndrome, caused by biallelic desmoglein 1 (DSG1) mutations, exhibit skin lesions reminiscent of erythrokeratodermia variabilis, caused by mutations in connexin (Cx) genes. We, therefore, examined whether SAM syndrome-causing DSG1 mutations interfere with Cx expression and GJ function. Lesional skin biopsies from SAM syndrome patients (n = 7) revealed decreased Dsg1 and Cx43 plasma membrane localization compared with control and nonlesional skin. Cultured keratinocytes and organotypic skin equivalents depleted of Dsg1 exhibited reduced Cx43 expression, rescued upon re-introduction of wild-type Dsg1, but not Dsg1 constructs modeling SAM syndrome-causing mutations. Ectopic Dsg1 expression increased cell-cell dye transfer, which Cx43 silencing inhibited, suggesting that Dsg1 promotes GJ function through Cx43. As GJA1 gene expression was not decreased upon Dsg1 loss, we hypothesized that Cx43 reduction was due to enhanced protein degradation. Supporting this, PKC-dependent Cx43 S368 phosphorylation, which signals Cx43 turnover, increased after Dsg1 depletion, while lysosomal inhibition restored Cx43 levels. These data reveal a role for Dsg1 in regulating epidermal Cx43 turnover.

Spiral steroids as potential markers for pre-eclampsia: A pilot study.

Pre-eclampsia is a condition that complicates 3-6% of pregnancies. At present, there is no FDA approved diagnostic method to evaluate risk or progression. We would like to report our observation of elevated levels of the spiral steroid phosphoester precursor in patients with pre-eclampsia. Samples from 20 normotensive pregnant women and from 20 women with pre-eclampsia were purchased from Global Alliance for the Prevention of Prematurity and Stillbirth (GAPPS). After addition of miltefosine (hexadecyl phosphocholine) as an internal control, each sample was extracted and evaluated by tandem MS. The method detected 2 spiral steroid phosphoesters and their common precursor. For each mass ion, the ion counts obtained were compared to the ion counts of miltefosine. The samples from the normotensive women were used to establish the mean and standard deviation. Then, Z-scores were determined for each of the serum components. Of the samples from the women with pre-eclampsia, 12 of 19 (63%) samples had Z-scores over 2.0 for at least one of the steroid phosphoesters. In contrast, current markers under development for risk of pre-eclampsia have prediction scores ranging from 8 to 33%. As the spiral steroids are lactones, similar to spironolactone and related compounds, they could function as endogenous potassium sparing hormones. However, as only about half of the affected patients had elevated levels of the spiral steroids, it may not be the only underlying pathology. Parturition would end transfer of placental spiral steroids and account for the termination of the symptoms of pre-eclampsia/eclampsia. Previous investigators have proposed inadequate placental function as the critical pathology of pre-eclampsia, but it is hard to imagine how inadequate placental function leads to the maternal pathology without invoking an endogenous potassium sparing hormone originating in the placenta.

Filaggrin 2 Deficiency Results in Abnormal Cell-Cell Adhesion in the Cornified Cell Layers and Causes Peeling Skin Syndrome Type A.

Peeling skin syndromes form a large and heterogeneous group of inherited disorders characterized by superficial detachment of the epidermal cornified cell layers, often associated with inflammatory features. Here we report on a consanguineous family featuring noninflammatory peeling of the skin exacerbated by exposure to heat and mechanical stress. Whole exome sequencing revealed a homozygous nonsense mutation in FLG2, encoding filaggrin 2, which cosegregated with the disease phenotype in the family. The mutation was found to result in decreased FLG2 RNA levels as well as almost total absence of filaggrin 2 in the patient epidermis. Filaggrin 2 was found to be expressed throughout the cornified cell layers and to colocalize with corneodesmosin that plays a crucial role in maintaining cell-cell adhesion in this region of the epidermis. The absence of filaggrin 2 in the patient skin was associated with markedly decreased corneodesmosin expression, which may contribute to the peeling phenotype displayed by the patients. Accordingly, using the dispase dissociation assay, we showed that FLG2 downregulation interferes with keratinocyte cell-cell adhesion. Of particular interest, this effect was aggravated by temperature elevation, consistent with the clinical phenotype. Restoration of corneodesmosin levels by ectopic expression rescued cell-cell adhesion. Taken together, the present data suggest that filaggrin 2 is essential for normal cell-cell adhesion in the cornified cell layers.

Calpain 12 Function Revealed through the Study of an Atypical Case of Autosomal Recessive Congenital Ichthyosis.

Congenital erythroderma is a rare and often life-threatening condition, which has been shown to result from mutations in several genes encoding important components of the epidermal differentiation program. Using whole exome sequencing, we identified in a child with congenital exfoliative erythroderma, hypotrichosis, severe nail dystrophy and failure to thrive, two heterozygous mutations in ABCA12 (c.2956C>T, p.R986W; c.5778+2T>C, p. G1900Mfs*16), a gene known to be associated with two forms of ichthyosis, autosomal recessive congenital ichthyosis, and harlequin ichthyosis. Because the patient displayed an atypical phenotype, including severe hair and nail manifestations, we scrutinized the exome sequencing data for additional potentially deleterious genetic variations in genes of relevance to the cornification process. Two mutations were identified in CAPN12, encoding a member of the calpain proteases: a paternal missense mutation (c.1511C>A; p.P504Q) and a maternal deletion due to activation of a cryptic splice site in exon 9 of the gene (c.1090_1129del; p.Val364Lysfs*11). The calpain 12 protein was found to be expressed in both the epidermis and hair follicle of normal skin, but its expression was dramatically reduced in the patient's skin. The downregulation of capn12 expression in zebrafish was associated with abnormal epidermal morphogenesis. Small interfering RNA knockdown of CAPN12 in three-dimensional human skin models was associated with acanthosis, disorganized epidermal architecture, and downregulation of several differentiation markers, including filaggrin. Accordingly, filaggrin expression was almost absent in the patient skin. Using ex vivo live imaging, small interfering RNA knockdown of calpain 12 in skin from K14-H2B GFP mice led to significant hair follicle catagen transformation compared with controls. In summary, our results indicate that calpain 12 plays an essential role during epidermal ontogenesis and normal hair follicle cycling and that its absence may aggravate the clinical manifestations of ABCA12 mutations.

SVEP1 plays a crucial role in epidermal differentiation.

SVEP1 is a recently identified multidomain cell adhesion protein, homologous to the mouse polydom protein, which has been shown to mediate cell-cell adhesion in an integrin-dependent manner in osteogenic cells. In this study, we characterized SVEP1 function in the epidermis. SVEP1 was found by qRT-PCR to be ubiquitously expressed in human tissues, including the skin. Confocal microscopy revealed that SVEP1 is normally mostly expressed in the cytoplasm of basal and suprabasal epidermal cells. Downregulation of SVEP1 expression in primary keratinocytes resulted in decreased expression of major epidermal differentiation markers. Similarly, SVEP1 downregulation was associated with disturbed differentiation and marked epidermal acanthosis in three-dimensional skin equivalents. In contrast, the dispase assay failed to demonstrate significant differences in adhesion between keratinocytes expressing normal vs low levels of SVEP1. Homozygous Svep1 knockout mice were embryonic lethal. Thus, to assess the importance of SVEP1 for normal skin homoeostasis in vivo, we downregulated SVEP1 in zebrafish embryos with a Svep1-specific splice morpholino. Scanning electron microscopy revealed a rugged epidermis with perturbed microridge formation in the centre of the keratinocytes of morphant larvae. Transmission electron microscopy analysis demonstrated abnormal epidermal cell-cell adhesion with disadhesion between cells in Svep1-deficient morphant larvae compared to controls. In summary, our results indicate that SVEP1 plays a critical role during epidermal differentiation.

Mutations in TSPEAR, Encoding a Regulator of Notch Signaling, Affect Tooth and Hair Follicle Morphogenesis.

Despite recent advances in our understanding of the pathogenesis of ectodermal dysplasias (EDs), the molecular basis of many of these disorders remains unknown. In the present study, we aimed at elucidating the genetic basis of a new form of ED featuring facial dysmorphism, scalp hypotrichosis and hypodontia. Using whole exome sequencing, we identified 2 frameshift and 2 missense mutations in TSPEAR segregating with the disease phenotype in 3 families. TSPEAR encodes the thrombospondin-type laminin G domain and EAR repeats (TSPEAR) protein, whose function is poorly understood. TSPEAR knock-down resulted in altered expression of genes known to be regulated by NOTCH and to be involved in murine hair and tooth development. Pathway analysis confirmed that down-regulation of TSPEAR in keratinocytes is likely to affect Notch signaling. Accordingly, using a luciferase-based reporter assay, we showed that TSPEAR knock-down is associated with decreased Notch signaling. In addition, NOTCH1 protein expression was reduced in patient scalp skin. Moreover, TSPEAR silencing in mouse hair follicle organ cultures was found to induce apoptosis in follicular epithelial cells, resulting in decreased hair bulb diameter. Collectively, these observations indicate that TSPEAR plays a critical, previously unrecognized role in human tooth and hair follicle morphogenesis through regulation of the Notch signaling pathway.

Identification of a Functional Risk Variant for Pemphigus Vulgaris in the ST18 Gene.

Pemphigus vulgaris (PV) is a life-threatening autoimmune mucocutaneous blistering disease caused by disruption of intercellular adhesion due to auto-antibodies directed against epithelial components. Treatment is limited to immunosuppressive agents, which are associated with serious adverse effects. The propensity to develop the disease is in part genetically determined. We therefore reasoned that the delineation of PV genetic basis may point to novel therapeutic strategies. Using a genome-wide association approach, we recently found that genetic variants in the vicinity of the ST18 gene confer a significant risk for the disease. Here, using targeted deep sequencing, we identified a PV-associated variant residing within the ST18 promoter region (p<0.0002; odds ratio = 2.03). This variant was found to drive increased gene transcription in a p53/p63-dependent manner, which may explain the fact that ST18 is up-regulated in the skin of PV patients. We then discovered that when overexpressed, ST18 stimulates PV serum-induced secretion of key inflammatory molecules and contributes to PV serum-induced disruption of keratinocyte cell-cell adhesion, two processes previously implicated in the pathogenesis of PV. Thus, the present findings indicate that ST18 may play a direct role in PV and consequently represents a potential target for the treatment of this disease.

Phosphatidylserine increases IKBKAP levels in a humanized knock-in IKBKAP mouse model.

Familial dysautonomia (FD) is a severe neurodegenerative genetic disorder restricted to the Ashkenazi Jewish population. The most common mutation in FD patients is a T-to-C transition at position 6 of intron 20 of the IKBKAP gene. This mutation causes aberrant skipping of exon 20 in a tissue-specific manner, leading to reduction of the IκB kinase complex-associated protein (IKAP) protein in the nervous system. We established a homozygous humanized mouse strain carrying human exon 20 and its two flanking introns; the 3' intron has the transition observed in the IKBKAP gene of FD patients. Although our FD humanized mouse does not display FD symptoms, the unique, tissue-specific splicing pattern of the IKBKAP in these mice allowed us to evaluate the effect of therapies on gene expression and exon 20 splicing. The FD mice were supplemented with phosphatidylserine (PS), a safe food supplement that increases mRNA and protein levels of IKBKAP in cell lines generated from FD patients. Here we demonstrated that PS treatment increases IKBAKP mRNA and IKAP protein levels in various tissues of FD mice without affecting exon 20 inclusion levels. We also observed that genes associated with transcription regulation and developmental processes were up-regulated in the cerebrum of PS-treated mice. Thus, PS holds promise for the treatment of FD.

Alternative approach to a heavy weight problem.

Obesity is reaching epidemic proportions in developed countries and represents a significant risk factor for hypertension, heart disease, diabetes, and dyslipidemia. Splicing mutations constitute at least 14% of disease-causing mutations, thus implicating polymorphisms that affect splicing as likely candidates for disease susceptibility. A recent study suggested that genes associated with obesity were significantly enriched for rare nucleotide variants. Here, we examined these variants and revealed that they are located near splice junctions and tend to affect exonic splicing regulatory sequences. We also show that the majority of the exons that harbor these SNPs are constitutively spliced, yet they exhibit weak splice sites, typical to alternatively spliced exons, and are hence suboptimal for recognition by the splicing machinery and prone to become alternatively spliced. Using ex vivo assays, we tested a few representative variants and show that they indeed affect splicing by causing a shift from a constitutive to an alternative pattern, suggesting a possible link between extreme body mass index and abnormal splicing patterns.

The catalytic properties of the recombinant reverse transcriptase of bovine immunodeficiency virus.

Bovine immunodeficiency virus (BIV) is a lentivirus with no proven pathogenesis in infected cattle. Yet, in experimentally infected rabbits, it causes an AIDS-like disease. Consequently, we expressed two recombinant isoforms of BIV reverse transcriptase (RT), which differ in their C-termini, and studied their catalytic properties. Both isoforms prefer Mg(+2) over Mn(+2) with most DNA polymerase and ribonuclease-H substrates. The processivity of DNA synthesis by the BIV RTs is higher than that of HIV-1 RT, whereas the fidelity of synthesis is even lower than that of the HIV-1 enzyme. The ribonuclease-H cleavage pattern suggests that the spatial distance between the polymerase and ribonuclease-H active sites of the two BIV RT isoforms equals 20 nt, unlike the 17 nt distance observed in almost all other RTs. The longer BIV RT version is somewhat less active than the shorter version, suggesting that the extra 74 residues (with homology to dUTPases) might obstruct efficient catalysis.